Dietary Docosahexaenoic Acid Reduces Oscillatory Wall Shear Stress, Atherosclerosis, and Hypertension, Most Likely Mediated via an IL‐1–Mediated Mechanism

Background Hypertension is a complex condition and a common cardiovascular risk factor. Dietary docosahexaenoic acid (DHA) modulates atherosclerosis and hypertension, possibly via an inflammatory mechanism. IL‐1 (interleukin 1) has an established role in atherosclerosis and inflammation, although whether IL‐1 inhibition modulates blood pressure is unclear. Methods and Results Male apoE−/− (apolipoprotein E–null) mice were fed either a high fat diet or a high fat diet plus DHA (300 mg/kg per day) for 12 weeks. Blood pressure and cardiac function were assessed, and effects of DHA on wall shear stress and atherosclerosis were determined. DHA supplementation improved left ventricular function, reduced wall shear stress and oscillatory shear at ostia in the descending aorta, and significantly lowered blood pressure compared with controls (119.5±7 versus 159.7±3 mm Hg, P<0.001, n=4 per group). Analysis of atheroma following DHA feeding in mice demonstrated a 4‐fold reduction in lesion burden in distal aortas and in brachiocephalic arteries (P<0.001, n=12 per group). In addition, DHA treatment selectively decreased plaque endothelial IL‐1β (P<0.01). Conclusions Our findings revealed that raised blood pressure can be reduced by inhibiting IL‐1 indirectly by administration of DHA in the diet through a mechanism that involves a reduction in wall shear stress and local expression of the proinflammatory cytokine IL‐1β.


Blood pressure analysis
Systolic and diastolic blood pressure was measured in the mice using a Visitech tailcuff system (Visitech Systems, NJ, USA) as described previously (32). Briefly, the mice were subjected to one week of training before starting the actual measurements in order to minimize stress levels. Blood pressure was measured in 4 mice per group, and 10 measurements per mouse per day were recorded for four different days per week for 12 weeks. The data were rejected if the systolic blood pressure was more than 200mmHg or less than 40mmHg, outside the 2SD of the mean or had fewer than 4 valid readings. Per week measurements were also rejected if they had fewer than 3 valid readings.

Echocardiography
To assess the effect of DHA feeding on cardiac function during the duration of the study, transthoracic echocardiogram (TE) was performed as previously described (33). Briefly, the mice (n=4/group) were anesthetised using isoflurane delivered in oxygen in an isolator before placing the mice in a supine position. To minimise excessive heat loss, which may affect the measurements, the mice were placed on a heated platform and continuous rectal temperature was monitored. During recording, the mice were maintained on approximately 0.5-1.5% (v/v) isoflurane in oxygen, delivered via a nose cone. The level of anesthesia was adjusted to achieve a heart rate close to 500 ± 125bpm. To allow smooth image acquisition, the mouse chest wall was depilated and ultrasound gel was applied (Aquasonics 100 Gel, Parker Labs Inc. New Jersey, U.S). Left ventricular (LV) function was recorded in the short axis view at the level of the mid-papillary muscles, whereas M-mode measurements were made for LV wall and cavity dimension (LVIDd). Ejection fraction (%EF), fractional shortening (%FS) and corrected LV mass (LVM) were assessed using automated analysis.
The blood was collected using cardiac puncture and plasma separated by centrifugation at 3000g over 5 minutes, collected and stored at -80 o C prior to analysis. The inflammatory markers (IL-6, IL-8, RANTES, and MCP) were measured in the plasma using a cytometric bead array (CBA; BD Bioscience) according to manufacturer's recommendations. Lipid profiles (total cholesterol, LDL and HDL/cholesterol ratio) were measured in the Clinical Biochemistry Laboratory at the Royal Hallamshire Hospital, Sheffield, UK.

Preparation of samples for erythrocyte fatty acid analysis
Freshly collected blood (by cardiac puncture) was collected into 4ml EDTA tubes (4 pooled samples of RBCs from 4 different mice per group with a minimum volume of 1mL). The blood samples were centrifuged for 5 minutes at 3000g and the plasma and buffy coat were aspirated and discarded. The tubes containing red cell fractions were then labelled and stored at -80 0 C until sent to BioLab, London, UK to conduct the analysis.

Atherosclerosis analysis
The extent of atherosclerosis was assessed in whole aortae by an en face method (35). In brief, the aortae were perfused firstly by PBS and then by 10% (v/v) formalin. After the exposure of the whole aortae and removal of the adherent fats and vessels, aortae were dissected from the levels of aortic valves to the aortic orifice of the diaphragm under a dissecting microscope. Aortae were then fixed in 10% (v/v) formalin overnight at 4 o C and stored in PBS at 4 o C until pinning was conducted. The aortae were opened longitudinally and stained with oil red O stain (ORS). For this, the aortae were rinsed in 60% (v/v) isopropanol and stained with 0.3% (w/v) Oil Red O (Sudan IV; Sigma O0625) in PBS for 30min followed by destaining for 20min in 60% (v/v) isopropanol and further washing in distilled water. The stained aortae were pinned on a wax filled petri dish (15cm) using micro-needles (Fine Science Tools, Heidelberg, Germany). Images were recorded using a digital camera connected to light microscope at 15x magnification. Lesion areas were analysed using a NIS-elements analysis software system (Nikon, UK). Atherosclerotic lesion in the whole aortae, arch and descending parts were quantified as % of the total surface area. A second assessment of atherosclerosis was conducted in cross-sectional aortic root and brachiocephalic sections as described (34). Briefly, the hearts and brachiocephalic arteries (first branch of the aortic arch) were collected and stored in 10% (v/v) formalin overnight at 4 o C and then in PBS until they were embedded in paraffin wax. The paraffin-embedded hearts (at the level of aortic valves) and brachiocephalic arteries were serially sectioned using a Leica RM2135 microtome (Leica Microsystems, Wetzlar, Germany). Sections (5μm thickness) were collected and stained with Alcian Blue & Elastic Van Gieson (AB/EVG) as described (30).

Analysis of Collagen content
Collagen content in aortic and brachiocephalic sections were measured in martius scarlet blue (MSB) positive stained areas as described (30) and expressed as a percentage of the total surface area. In brief, the tissues were dewaxed and rehydrated in xylene and graded alcohols. The tissues were then stained with 1% (w/v) Celestine blue for 5 minutes, drained, and stained with Harris' haematoxylin for 5 minutes.